Myelin Oligodendrocyte Glycoprotein Epitope Research Articles

Pathogenesis, Clinical Features, and Treatment of Patients with Myelin Oligodendrocyte Glycoprotein (MOG) Autoantibody-Associated Disorders Focusing on Optic Neuritis with Consideration of Autoantibody-Binding Sites: A Review.

Although there is a substantial amount of data on the clinical characteristics, diagnostic criteria, and pathogenesis of myelin oligodendrocyte glycoprotein (MOG) autoantibody-associated disease (MOGAD), there is still uncertainty regarding the MOG protein function and the pathogenicity of anti-MOG autoantibodies in this disease. It is important to note that the disease characteristics, immunopathology, and treatment response of MOGAD patients differ from those of anti-aquaporin 4 antibody-positive neuromyelitis optica spectrum disorders (NMOSDs) and multiple sclerosis (MS). The clinical phenotypes of MOGAD are varied and can include acute disseminated encephalomyelitis, transverse myelitis, cerebral cortical encephalitis, brainstem or cerebellar symptoms, and optic neuritis. The frequency of optic neuritis suggests that the optic nerve is the most vulnerable lesion in MOGAD. During the acute stage, the optic nerve shows significant swelling with severe visual symptoms, and an MRI of the optic nerve and brain lesion tends to show an edematous appearance. These features can be alleviated with early extensive immune therapy, which may suggest that the initial attack of anti-MOG autoantibodies could target the structures on the blood-brain barrier or vessel membrane before reaching MOG protein on myelin or oligodendrocytes. To understand the pathogenesis of MOGAD, proper animal models are crucial. However, anti-MOG autoantibodies isolated from patients with MOGAD do not recognize mouse MOG efficiently. Several studies have identified two MOG epitopes that exhibit strong affinity with human anti-MOG autoantibodies, particularly those isolated from patients with the optic neuritis phenotype. Nonetheless, the relations between epitopes on MOG protein remain unclear and need to be identified in the future.

Competitive ELISA for the identification of 35-55 myelin oligodendrocyte glycoprotein immunodominant epitope conjugated with mannan.

Analogs of immunodominant myelin peptides involved in multiple sclerosis (MS: the most common autoimmune disease) have been extensively used to modify the immune response over the progression of the disease. The immunodominant 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG35-55 ) is an autoantigen appearing in MS and stimulates the encephalitogenic T cells, whereas mannan polysaccharide (Saccharomyces cerevisiae) is a carrier toward the mannose receptor of dendritic cells and macrophages. The conjugate of mannan-MOG35-55 has been extensively studied for the inhibition of chronic experimental autoimmune encephalomyelitis (EAE: an animal model of MS) by inducing antigen-specific immune tolerance against the clinical symptoms of EAE in mice. Moreover, it presents a promising approach for the immunotherapy of MS under clinical investigation. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the MOG35-55 peptide that is conjugated to mannan. Intra- and inter-day assay experiments proved that the proposed ELISA methodology is accurate and reliable and could be used in the following applications: (i) to identify the peptide (antigen) while it is conjugated to mannan and (ii) to adequately address the alterations that the MOG35-55 peptide may undergo when it is bound to mannan during production and stability studies.

Development of PLGA Nanoparticles with a Glycosylated Myelin Oligodendrocyte Glycoprotein Epitope (MOG35-55) against Experimental Autoimmune Encephalomyelitis (EAE).

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases in young adults, with early clinical symptoms seen in the central nervous system (CNS) myelin sheaths due to an attack caused by the patient's immune system. Activation of the immune system is mediated by the induction of an antigen-specific immune response involving the interaction of multiple T-cell types with antigen-presenting cells (APCs), such as dendritic cells (DCs). Antigen-specific therapeutic approaches focus on immune cells and autoantigens involved in the onset of disease symptoms, which are the main components of myelin proteins. The ability of such therapeutics to bind strongly to DCs could lead to immune system tolerance to the disease. Many modern approaches are based on peptide-based research, as, in recent years, they have been of particular interest in the development of new pharmaceuticals. The characteristics of peptides, such as short lifespan in the body and rapid hydrolysis, can be overcome by their entrapment in nanospheres, providing better pharmacokinetics and bioavailability. The present study describes the development of polymeric nanoparticles with encapsulated myelin peptide analogues involved in the development of MS, along with their biological evaluation as inhibitors of MS development and progression. In particular, particles of poly(lactic-co-glycolic) acid (PLGA) loaded with peptides based on mouse/rat (rMOG) epitope 35-55 of myelin oligodendrocyte glycoprotein (MOG) conjugated with saccharide residues were developed. More specifically, the MOG35-55 peptide was conjugated with glucosamine to promote the interaction with mannose receptors (MRs) expressed by DCs. In addition, a study of slow release (dissolution) and quantification on both initially encapsulated peptide and daily release in saline in vitro was performed, followed by an evaluation of in vivo activity of the formulation on mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS, using both prophylactic and therapeutic protocols. Our results showed that the therapeutic protocol was effective in reducing EAE clinical scores and inflammation of the central nervous system and could be an alternative and promising approach against MS inducing tolerance against the disease.

MOG variant 45D does not properly activate high-affinity, MOG reactive effector T cells but allows for high-affinity FoxP3+ Treg function

Abstract Although it’s well appreciated that autoreactive T cells can drive autoimmune disease, how T cells precisely distinguish and respond to epitopes is unclear. For example, T cells can proliferate in response to multiple epitopes of myelin oligodendrocyte glycoprotein (MOG), but not all epitopes are encephalitogenic. Here, we investigate why T cells specific for MOG 35–55 are encephalitogenic. We compare the wildtype MOG epitope (wtMOG) to a single amino acid substitute, 45D, which was engineered to expresses the same T cell receptor (TCR) contact residues as wtMOG but is mutated such that its affinity for MHC II is decreased. We employed the micropipette adhesion frequency assay to demonstrate that a monoclonal, MOG-specific, high-affinity TCR binds to 45D with the same affinity as wtMOG. Despite conserved TCR contact residues, the MOG 45D variant is not encephalitogenic and fails to prime high-affinity tetramer-positive TCR clones. We also extended our polyclonal analysis to FoxP3+ regulatory T cells (Tregs) and found that, compared to wtMOG, 45D priming increases the frequency of MOG-specific Tregs. We further show that these Tregs are functional and suppress the proliferation of MOG-specific T cells. Our work begins to dissect the differential T cell receptor kinetic properties that allow for a response, or lack thereof, in Tregs and Teffs to myelin peptides. Supported by NIH R01 NS071518-09

Conserved spinal cord bioenergetics in experimental autoimmune encephalomyelitis in C57BL6 mice, measured using phosphorescence oxygen analyzer

BackgroundWe have previously reported that spinal cord respiration (cellular mitochondrial oxygen consumption) and ATP content are conserved in the studied model of experimental autoimmune encephalomyelitis (EAE), foreseeing a recovery of the diseased rats. This exemplary lesion of multiple sclerosis is used here to measure spinal cord bioenergetics in C57BL6 mice. Our hypothesis is that, despite the well-known focal axonal mitochondrial pathology, bioenergetics of the CNS is reasonably preserved in this disease. MethodsEAE was induced with an immunodominant myelin oligodendrocyte glycoprotein epitope in complete Freund's adjuvant, appended by injections of pertussis toxin. A low- and high-dose of the encephalitogen, administered into base of tail or hind-flank, were investigated. Control mice received only the incomplete adjuvant into tail. Oxygen measurements were based on quenching the phosphorescence of Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin by molecular oxygen. Cellular ATP was measured using the luciferin/luciferase system. ResultsThe kinetics of spinal cord oxygen consumption was zero-order (linear with time) and inhibited by cyanide, confirming oxygen was reduced by cytochrome oxidase. The rate of respiration (in μM O2.min−1.mg−1; measured on Days 13–28) in control mice was (mean ± SD) 0.086 ± 0.024 (n = 8) and in immunized mice was 0.079 ± 0.020 (n = 15, P = 0.265, Mann-Whitney test). Consistently, cellular ATP (in μmol mg−1 dry pellet weight; measured on Days 13–28) in control mice was 0.068 ± 0.079 (n = 11) and in immunized mice was 0.063 ± 0.061 (n = 24, P = 0.887, Mann-Whitney U test). ConclusionsIn vitro measurements of spinal cord bioenergetics show conservation of the mitochondrial function in mice with EAE. These results suggest the previously documented reduced mitochondrial electrochemical potential in this disease is alterable, and likely reflects the adverse events of neuroinflammation.

Induction with MHC variant peptide MOG 45D generates MOG-reactive T cells but no clinical disease

Abstract Demyelinating disease can be driven by CD4+ T cells specific for myelin oligodendrocyte glycoprotein (MOG). We have previously decreased the affinity of MOG for Class II MHC by mutating the P6 anchor residue from an S to a D amino acid, creating the 45D MHC variant peptide. 45D is incapable of inducing experimental autoimmune encephalomyelitis (EAE) despite its CD4 epitope bearing the same T cell receptor contact residues as the wildtype MOG epitope (wtMOG). Although the wtMOG and 45D peptides generate CD4 T cells specific for MOG in the periphery and CNS, there is an overall decrease in the number of high-affinity CD4 T cells in response to 45D priming. Analysis of the responding T cell populations to 45D also found increases in MOG-reactive Tregs and decreases in MOG-specific CD8 T cells. Thus, the variant peptide 45D alters the MOG-specific T cell response in favor of peripheral tolerance mechanisms that ultimately result in the absence of clinical disease.

Archeological neuroimmunology: resurrection of a pathogenic immune response from a historical case sheds light on human autoimmune encephalomyelitis and multiple sclerosis

Aim of our study was to identify the target auto-antigen in the central nervous system recognized by the immune system of a unique patient, who died more than 60 years ago from a disease with pathological changes closely resembling multiple sclerosis (MS), following a misguided immunization with lyophilized calf brain tissue. Total mRNA was isolated from formaldehyde fixed and paraffin embedded archival brain tissue containing chronic active inflammatory demyelinating lesions with inflammatory infiltrates rich in B-lymphocytes and plasma cells. Analysis of the transcriptome by next generation sequencing and reconstruction of the dominant antibody by bioinformatic tools revealed the presence of one strongly expanded B-cell clone, producing an autoantibody against a conformational epitope of myelin oligodendrocytes glycoprotein (MOG), similar to that recognized by the well characterized monoclonal anti-MOG antibody 8-18C5. The reconstructed antibody induced demyelination after systemic or intrathecal injection into animals with T-cell mediated encephalomyelitis. Our study suggests that immunization with bovine brain tissue in humans may—in a small subset of patients—induce a disease with an intermediate clinical and pathological presentation between MS and MOG-antibody associated inflammatory demyelinating disease (MOGAD).

Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein

Abstract Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the role B cells play in pathogenesis and regulation of disease. Historical dissection of B cell function in the MS mouse model experimental autoimmune encephalomyelitis (EAE) has largely been confined to disease induced with either the immunodominant epitope of myelin oligodendrocyte glycoprotein (MOG35–55) or full length recombinant human MOG protein (hrMOG), the latter representing the only available B cell-dependent EAE model. We have demonstrated that myelin proteolipid protein (PLP178–191)-specific CD8 T cells possess striking EAE-suppression capacity. However, little is known regarding the role of B cells in these contexts. Interestingly, unlike MOG35–55, where genetic absence or depletion of B cells yields a more severe disease course, PLP178–191 yields identical EAE progression in both WT and μMT mice, suggesting suboptimal B cell engagement. Given the reports that hrMOG drives a B cell-dependent demyelinating disease versus its MOG35–55 counterpart, we hypothesized that a longer PLP antigen consisting of more epitopes may better engage B cells and provide an additional model for the field. To this end, we designed a novel peptide encompassing the extracellular domains (ECD) of PLP and compared its immunogenicity and disease-inducing capacity with PLP178–191 in μMT and WT mice. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE disease compared to WT counterparts. PLPECD also induced EAE in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.

Investigating the antigen-specific T cell response to MHC variant peptide 45D

Abstract We have previously generated a variant of the myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide by mutation of the P6 MHC anchor residue from an S to a D amino acid. Here, we confirm previous findings that the 45D MHC variant peptide is incapable of inducing experimental autoimmune encephalomyelitis (EAE) despite its CD4 epitope bearing the same TCR contact residues as wildtype MOG epitope (wtMOG). Using the micropipette adhesion frequency assay (MP), we show that induction with 45D generates reduced levels of CD4 T cells specific for MOG in the CNS but not the periphery of asymptomatic mice. Importantly, 45D induction results in a loss of high affinity CD4 T cells, which could be responsible for initiation of disease. We also found changes in MOG-reactivity within the peripheral Treg compartment of 45D-induced mice. Together, the variant peptide alters the MOG-specific Teff and Treg response. Although the 45D mutation was designed to target the CD4 MOG epitope, it also changes the antigen responsible for expanding MOG-reactive CD8 T cells. We find that priming with 45D expands CD8 T cells with heightened affinity for MOG. Taken together, our data demonstrate that 45D may enhance peripheral tolerance mechanisms that ultimately aid in the protection from autoimmune disease.

In silico study of the association of the HLA-A*31:01 allele (human leucocyte antigen allele 31:01) with neuroantigenic epitopes of PLP (proteolipid protein), MBP (myelin basic protein) and MOG proteins (myelin oligodendrocyte glycoprotein) for studying the multiple sclerosis disease pathogenesis

The main pathologic hallmark of multiple sclerosis is a demyelinating plaque that contains a prominent immunologic response dominated by T cells of the immune system. PLP (proteolipid protein), MPB (myelin basic protein), and Myelin oligodendrocyte glycoprotein (MOG) proteins are important autoantigens for the demyelinating of CNS in multiple sclerosis. There is good evidence indicating that T CD8+ cells and MHC class I molecules play an important role in this disease. The HLA-A*31:01 allele of MHC class I is a member of HLA-A3 superfamily and there is no clear report concerning the relationship of this allele with MS. Feeling this gap, we studied the possible association of the HLA-A*31:01 with MS by prediction of neuroantigenic epitopes of human MBP, PLP, and MOG proteins of myelin sheath using in silico methods. PLP did not show any neuroantigenic epitope, but the two epitopes of MBP and seven epitopes of MOG for HLA-A*31:01 were determined via bioinformatics servers. In silico study of the nine epitope showed that MOG195-204 (LIICYNWLHR) peptide of the membrane-associated/cytoplasmic part of human MOG has suitable binding affinity to the HLA-A*31:01 allele as a potential neuroantigenic epitope. Further investigations of this peptide revealed that the binding of C-terminal residue of this peptide has a more significant effect on binding to this allele than the N-terminal part of the peptide. Altogether, this combination of “LIICYNWLHR/A*31:01 allele “may play an important role in MS pathogenesis and this complex is suggested for further studies such as T cell receptor. Communicated by Ramaswamy H. Sarma

Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein

Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the pathogenic and regulatory roles these cells play in demyelinating disease. Dissecting the function of B cells in the MS mouse model experimental autoimmune encephalomyelitis (EAE) is largely confined to induction with either the myelin oligodendrocyte glycoprotein epitope MOG35–55 or the full-length recombinant human MOG protein, the latter representing the most-used B cell-dependent EAE model. There is a clear need to investigate B cell function in additional myelin antigen contexts. Unlike MOG35–55, where lack of B cells yields more severe disease, we show here that the immunodominant myelin proteolipid protein epitope (PLP178–191) elicited identical EAE in WT and μMT mice, suggesting an absence of B cell engagement by this peptide. We hypothesized that a longer PLP antigen may better engage B cells and designed a peptide encompassing the extracellular domains (ECD) of PLP. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE. In contrast, PLPECD induced EAE not only in WT mice, but in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B cell-dependent EAE model.

Identification of circulating MOG-specific B cells in patients with MOG antibodies.

ObjectiveTo identify circulating myelin oligodendrocyte glycoprotein (MOG)-specific B cells in the blood of patients with MOG antibodies (Abs) and to determine whether circulating MOG-specific B cells are linked to levels and epitope specificity of serum anti-MOG-Abs.MethodsWe compared peripheral blood from 21 patients with MOG-Abs and 26 controls for the presence of MOG-specific B cells. We differentiated blood-derived B cells in vitro in separate culture wells to Ab-producing cells via engagement of Toll-like receptors 7 and 8. We quantified the anti-MOG reactivity with a live cell–based assay by flow cytometry. We determined the recognition of MOG epitopes with a panel of mutated variants of MOG.ResultsMOG-Ab–positive patients had a higher frequency of MOG-specific B cells in blood than controls, but MOG-specific B cells were only detected in about 60% of these patients. MOG-specific B cells in blood showed no correlation with anti-MOG Ab levels in serum, neither in the whole group nor in the untreated patients. Epitope analysis of MOG-Abs secreted from MOG-specific B cells cultured in different wells revealed an intraindividual heterogeneity of the anti-MOG autoimmunity.ConclusionsThis study shows that patients with MOG-Abs greatly differ in the abundance of circulating MOG-specific B cells, which are not linked to levels of MOG-Abs in serum suggesting different sources of MOG-Abs. Identification of MOG-specific B cells in blood could be of future relevance for selecting patients with MOG-Abs for B cell–directed therapy.

Cross-reactivity between myelin oligodendrocyte glycoprotein and human endogenous retrovirus W protein: nanotechnological evidence for the potential trigger of multiple sclerosis

Multiple sclerosis (MS) is an autoimmune and inflammatory demyelinating disease of the central nervous system. Experimental evidence supports the reactivity of autoantibodies against components of myelin sheath including the myelin oligodendrocyte glycoprotein (MOG). The MS etiology is still unknown, but some risk factors associated with immune dysregulation, genetic susceptibility, and environmental factors are under investigation. The last consider the hypothesis of molecular mimicry mechanism, which is potentially triggered by viral antigen inducing MS autoimmunity. The Human Endogenous Retroviruses W family (HERV-W) is the subject of studies within this field, based on the detection of HERV-W envelope gene proteins in MS patients' samples. In the biomedical field of diagnosis and therapeutics, nanotechnology is of great use for the detailed study of molecular mechanisms involving specific interactions between biomolecules providing high specificity and sensitivity of response. In view of the significance of etiological aspects for the comprehension of MS mechanisms of action, we applied a nanotechnological approach designed for antibody detection. For this, we analyzed MOG peptide sequences similar to the HERV-W protein. These sequences were subjected to interaction with anti-HERV-W antibodies using atomic force spectroscopy (AFS) and silver nanoparticles (AgNPs) methods to survey the potential occurrence of molecular mimicry. Our results revealed the molecular recognition between the anti-HERV-W antibody and the HERV-W and MOG epitopes by AFS and AgNPs approaches. Specific non-linear shape of force curves and median adhesion force values within the expected range for an antigen-antibody interaction were obtained for HERV-W and MOG peptides, 163 pN and 178 pN, respectively, suggesting the occurrence of cross-reactivity in these systems.

Design and molecular dynamic simulation of a new double-epitope tolerogenic protein as a potential vaccine for multiple sclerosis disease.

One of the debilitating diseases affecting the central nervous system is multiple sclerosis (MS). As there is no definitive treatment for MS, researchers have mainly consented with optimization of strategies which slows down the progression of the disease such as specific auto-antigens tolerance induction. In this regard, the aim of this study was design of a new double-epitope protective vaccine based on interleukin (IL)-16-neuroantigens fusion proteins. First, we selected highly antigenic epitopes of myelin basic protein (MBP) (aa 84-104) and myelin oligodendrocyte glycoprotein (MOG) (aa 99-107) from available literature and our bioinformatics analysis. The correct cleavage of our constructs and major histocompatibility complex class II binding affinities of cleaved epitopes were checked and evaluated using Pepcleave and IEDB servers, respectively. Then, different combination of MOG and MBP epitopes with or without fusion to C-terminal active part of IL-16 were designed as constructs. Afterward, Modeller and Gromacs softwares used for the investigation of the MBP, and MOG epitopes antigenicity in these constructs. The results of molecular dynamics simulations showed that IL-16 in MOG + linker + MBP + IL-16 construct does not interfere with final epitopes antigenicity of MOG + linker + MBP construct. To sum up, the construct with IL-16 is suggested as a new double-epitope tolerogenic vaccine for prevention and amelioration of MS in human.

Pathogenicity of human antibodies against myelin oligodendrocyte glycoprotein.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with inflammatory demyelinating diseases of the central nervous system (CNS). We analyzed their pathogenic activity by affinity-purifying these antibodies (Abs) from patients and transferring them to experimental animals. Patients with Abs to MOG were identified by cell-based assay. We determined the cross-reactivity to rodent MOG and the recognized MOG epitopes. We produced the correctly folded extracellular domain of MOG and affinity-purified MOG-specific Abs from the blood of patients. These purified Abs were used to stain CNS tissue and transferred in 2 models of experimental autoimmune encephalomyelitis. Animals were analyzed histopathologically. We identified 17 patients with MOG Abs from our outpatient clinic and selected 2 with a cross-reactivity to rodent MOG; both had recurrent optic neuritis. Affinity-purified Abs recognized MOG on transfected cells and stained myelin in tissue sections. The Abs from the 2 patients recognized different epitopes on MOG, the CC' and the FG loop. In both patients, these Abs persisted during our observation period of 2 to 3 years. The anti-MOG Abs from both patients were pathogenic upon intrathecal injection in 2 different rat models. Together with cognate MOG-specific T cells, these Abs enhanced T-cell infiltration; together with myelin basic protein-specific T cells, they induced demyelination associated with deposition of C9neo, resembling a multiple sclerosis type II pathology. MOG-specific Abs affinity purified from patients with inflammatory demyelinating disease induce pathological changes in vivo upon cotransfer with myelin-reactive T cells, suggesting that these Abs are similarly pathogenic in patients. Ann Neurol 2018;84:315-328.

Experimental Autoimmune Encephalomyelitis (EAE) Model of Cynomolgus Macaques Induced by Recombinant Human MOG1-125 (rhMOG1-125) Protein and MOG34-56 Peptide.

Experimental autoimmune encephalomyelitis (EAE) induced by self-myelin antigen is a widely used in multiple sclerosis (MS) model for preclinical studies of new therapeutics and potential pathogenesis. By comparison with rodent EAE models, EAE models in primates are more similar to MS. Some groups have developed EAE models in primates by using common marmoset (Callithrix jacchus). However, this model has some limitations. EAE in cynomolgus monkey (Macaque fasciculrais) could overcome these limitations. The main objective of this study was to establish an ideal EAE cynomolgus monkey model resembling human MS by immunizing human myelin oligodendrocyte glycoprotein (MOG) protein. In this study, six female cynomolgus monkeys were divided into two separate experiment groups and one monkey as the control. EAE was induced in cynomolgus monkey by immunizing with the recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1-125) (rhMOG1-125) and a synthetic peptide, representing peptide 34-56 of human myelin oligodendrocyte glycoprotein (MOG34-56) in complete Freund's adjuvant (CFA) by subcutaneous multipoint immunization in the armpit and inguinal region and in combination with intravenous injection of pertussis toxin, and subsequent booster immunizations with the same dose of antigen in incomplete Freund's adjuvant (IFA) until the animals developed clinically significant EAE (score≥2). The body weight, clinical scores, magnetic resonance imaging (MRI), histopathology, antibody, cytokine profiling and antigen-specific lymphocyte proliferation were evaluated. The results showed that despite the different time intervals between onset and significant neurological deficits, all the cynomolgus monkeys immunized with rhMOG1-125 or MOG34-56 developed clinically significant acute fulminant or mild forms of EAE, with a success rate of 100%. The clinical courses were obvious heterogeneity which closely resembles MS. MOG34-56 immunization was much milder compared with rhMOG1-125 immunized individuals, at least in cynomolgus monkeys. Inflammation and demyelinated lesions were present in the brains and spinal cords. Immune profiling revealed high IgG levels associated with early onset of EAE but not the course of the disease. Significant antigen-specific T lymphocyte responses against immunodominant epitopes of MOG were also detected. Our results indicated rhMOG1-125 and MOG34-56 could induce successfully EAE models resembling MS in cynomolgus monkeys. The synergistic action of anti-myelin T cells and Abs during the pathogenesis of EAE could establish a more ideal EAE model.

NFM Cross-Reactivity to MOG Does Not Expand a Critical Threshold Level of High-Affinity T Cells Necessary for Onset of Demyelinating Disease.

Of interest to the etiology of demyelinating autoimmune disease is the potential to aberrantly activate CD4+ T cells due to cross-recognition of multiple self-epitopes such as has been suggested for myelin oligodendrocyte glycoprotein epitope 35-55 (MOG35-55) and neurofilament medium protein epitope 15-35 (NFM15-35). NFM15-35 is immunogenic in C57BL/6 mice but fails to induce demyelinating disease by polyclonal T cells despite having the same TCR contact residues as MOG35-55, a known encephalitogenic Ag. Despite reported cross-reactivity with MOG-specific T cells, the polyclonal response to NFM15-35 did not expand threshold numbers of MOG38-49 tetramer-positive T cells. Furthermore, NFM lacked functional synergy with MOG to promote experimental autoimmune encephalomyelitis because NFM-deficient synonymous with knockout mice developed an identical disease course to wild-type mice after challenge with MOG35-55 Single-cell analysis of encephalitogenic T cells using the peptide:MHC monomer-based two-dimensional micropipette adhesion frequency assay confirmed that NFM was not a critical Ag driving demyelinating disease because NFM18-30-specific T cells in the CNS were predominantly reactive to MOG38-49 The absence of NFM contribution to disease allowed mapping of the amino acids required for encephalitogenicity and expansion of high-affinity, MOG-specific T cells that defined the polyclonal response. Alterations of N-terminal residues outside of the NFM15-35 core nonamer promoted expansion of high-affinity, MOG38-49 tetramer-positive T cells and promoted consistent experimental autoimmune encephalomyelitis induction, unlike mice challenged with NFM15-35 Although NFM15-35 is immunogenic and cross-reactive with MOG at the polyclonal level, it fails to expand a threshold level of encephalitogenic, high-affinity MOG-specific T cells.

Recovery and quantification of a myelin oligodendrocyte glycoprotein peptide from rat plasma after protein precipitation

The recovery of high molecular weight peptides from complex biological samples is a challenging task. Herein, a reliable, cost effective and rapid methodology was developed for the recovery and quantification of a myelin oligodendrocyte glycoprotein epitope namely (LysGly)5MOG35-55, from rat plasma. Removal of plasma proteins before quantification of the peptide was achieved after precipitation by an acetonitrile/water/formic acid solution. Using the developed protocol, average recoveries of the peptide from plasma ranged between 83.3 and 90.3%.

Anemoside A3 ameliorates experimental autoimmune encephalomyelitis by modulating T helper 17 cell response.

Anemoside A3 (AA3) is a natural triterpenoid glycoside isolated from the root of Pulsatilla chinensis (Bunge) Regel. We previously showed that AA3 exhibits cognitive-enhancing and neuroprotective properties. In the present study, we demonstrated that AA3 modulates inflammatory responses by regulating prostaglandin E receptor 4 signaling. Because prostaglandin E receptor 4 is involved in the pathophysiology of experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), we assessed the beneficial effect of AA3 in EAE mice. AA3 treatment significantly reduced clinical severity and inflammatory infiltrates in the spinal cord of EAE mice. In vitro studies revealed that AA3 inhibited the T cell response toward the encephalitogenic epitope of myelin oligodendrocyte glycoprotein (MOG). AA3 significantly downregulated the expressions of certain Th1 and Th17 cytokines in activated T cells re-stimulated by MOG. Moreover, AA3 inhibited the activation of STAT4 and STAT3, which are the transcription factors pivotal for Th1 and Th17 lineage differentiation, respectively, in activated T cells. Pharmacological analysis further suggested that AA3 reduced Th17 cell differentiation and expansion. In conclusion, AA3 exerts an immunomodulatory effect in EAE, demonstrating its potential as a therapeutic agent for MS in humans.

A dynamic approach for treating autoimmune disease.

Abstract Cell-based immunotherapies have the potential to reverse autoimmune disease by inducing or restoring immune-tolerance. Extensive preclinical studies have demonstrated that regulatory T cell (Tregs) play a key role in both induction and maintenance of tolerance. Several strategies aimed at harvesting and expanding autologous Tregs and then re-infusing them into patients have been investigated. Unfortunately, development of a large scale in vitro method has not been achieved, especially for human cells. On the other hand, a clinically relevant in vivo approach for inducing Ag-specific tolerance has successfully been demonstrated using ectopic expression of an antigen in the liver. Here, we have developed a long-lasting immunotherapy that induces functionally suppressive, Ag-specific Tregs in the murine model of multiple sclerosis using a liver directed adeno-associated virus (AAV) vector expressing myelin oligodendrocyte glycoprotein (MOG). Modulation of EAE disease in C57BL/6 is typically limited to a single immunodominant epitope. By engineering the AAV vector to express full-length protein (MOG1-247), we have created an immunotherapy that is capable of dynamically inducing Ag-specific tolerance against multiple immunogenic epitopes (e.g MOG35-55, MOG119-132). Mice received a single peripheral injection of AAV.MOG vector, or control. EAE was induced between 2 weeks and 8 months later using various MOG epitopes emulsified in adjuvant. Within 14-days, control mice developed significant neurological deficits, whereas none of the treated mice exhibited any symptoms. This work completely abrogates the deficiencies associated with ex vivo expanded Tregs providing a new paradigm for treating autoimmune diseases.